Acetylation of lysine residues is a widespread protein post-translational modification (PTM), and extensively relevant to modulation of cellular processes, including protein conformation and interaction (Science. 2009 325: 834-840). Histone lysine acetylation was historically proposed to be a hallmark of transcriptionally active genes (Bioessays. 1998; 20:615-626), and hitherto, deregulation of histone acetylation patterns often drives the aberrant expression of oncogenes resulting in proliferation and tumorigenesis (Curr Opin Struct Biol. 2011 21: 735-743). Three types of proteins have been identified to regulate lysine acetylation: bromodomain (BRD) proteins (Nat Rev Drug Discov. 2014 13: 337-356), histone acetyltransferases (HATs), histone deacetylases (HDACs) and sirtuins (SIRTs) (Cell Death Dis. 2014 5: e1047). BRD proteins bind to acetylated lysine (Kac) and thus acting as readers of lysine acetylation state; HATs effect lysine acetylation acting as writers; HDACs and SIRTs remove acetyl groups as erasers (Cell. 2013 154: 569-582). Bromodomains, functioning as acetyl-lysine binding domains, belong to a family of evolutionarily conserved protein modules originally found in proteins associated with chromatin and in nearly all nuclear HATs (Biochim Biophys Acta. 2014 8:676-685). BRDs may contribute to highly specific histone acetylation by tethering transcriptional HATs to specific chromosomal sites, or to the activity of multi-protein complexes in chromatin remodeling. Thus, BRDs modulate enzyme activities, protein assembly and protein-protein interactions (PPIs) via lysine acetylation, revealing broad implications for the mechanisms underlying a wide variety of cellular events, such as transcriptional activation and chromatin remodeling.
Human genome encodes 61 BRDs in 46 different proteins, in which legend specificity is imparted in the amino acid residue differences around the acetyllysine binding site (FEBS Lett. 2012 586: 2692-2704). BRD proteins mostly contain one or two bromodomains, while some proteins, suchm as nuclear scaffolding proteins (PB1), contain more than two BRDs (Cell. 2013 153: 320-334). Bromodomain and extra-terminal (BET), which taxonomically belongs to human BRD proteins family, shares a common domain architecture comprising two N-terminal bromodomains and an extra-C terminal domain.
Bromodomains are small (about 110 amino acid) distinct domains within proteins that bind to acetylated lysine resides commonly but not exclusively in the context of histones. There is a family of around 50 proteins known to contain bromodomains, and they have a range of functions within the cell. The BET (bromodomain and extra-terminal) family of bromodomain containing proteins comprises 4 proteins (BRD2, BRD3, BRD4 and BRD-T) which contain tandem bromo domains capable of binding to two acetylated lysine residues in close proximity, increasing the specificity of the interaction. Recent research has established a compelling rationale for targeting BRD4 in cancer. BRD4 remains bound to transcriptional start sites of genes expressed during the entry into the G 1 phase of the cell cycle, and is functioning to recruit the positive transcription elongation factor complex (P-TEFb), resulting in increased expression of growth promoting genes (Mol. Cell. Biol. 2008 28, 967).
Except BRDT specially locating in testis, BET proteins are widely distributed, and exert function to regulate an array of cellular processes. Firstly detected as protein scaffolds, BET family proteins recruit variety proteins to chromatin and transcription sites. During interphase, BRD4 recruits positive transcriptional elongation factor complex (PTEFb) to sites of active transcription, while another pool of BRD4 may be recruited by transcription mediator complexes independent of PTEFb (J Biol Chem. 2007 13141-13145). In addition, the ET domain of BRD4 independently recruits transcription-modifying factors, including glioma tumor suppressor candidate region gene 1 (GLTSCR1); NSD3, a SET domain-containing histone methyltransferase; JMJD6, a histone arginine demethylase; and CHD4, a catalytic component of the NuRD nucleosome remodeling complex.
BET family may also function as mitotic bookmark, identifying actively transcribed genes during mitosis by remaining associated chromatin when all the other factors dissociate. BRD4 remains associated with H4K5ac histones on chromatin during mitosis, leading to rapid de-compaction of the surrounding chromatin and to transcription post-mitotically [Transcription. 2013 4 1: 13-17). BRD4 marks the start sites of many M/G1 genes, and accelerates expression of G1 genes and promotes cell cycle progression to S phase (Nat Cell Biol. 2011; 13:1295-1304). BRD4 seems to be required for the G2 to M phase transition of the cell cycle because microinjection of BRD4-specific antibodies leads to cell cycle arrest. Then, BET family proteins function as cell cycle regulators, as mentioned before, that key transcriptional regulators genes of S phase, E2F1 and E2F2, are associated with BRD2 multi-protein complexes. BRD3-dependent functional relationships with the cell cycle control machinery in normal cells are poorly understood, although forced expression of BRD3 down-regulates the RB-E2F pathway in nasopharyngeal carcinoma cells (Nature Reviews Cancer 2012, 12, 7: 465-477).
Importantly, BRD4 has been identified as a component of a recurrent t(15; 19) chromosomal translocation in an aggressive form of human squamous carcinoma (Cancer Res. 2003 63, 304,). Such translocations express the tandem N-terminal bromo domains of BRD4 as an in-frame chimera with the NUT (nuclear protein in testis) protein, genetically defining the so-called NUT midline carcinoma (NMC). Functional studies in patient-derived NMC cell lines have validated the essential role of the BRD4-NUT oncoprotein in maintaining the proliferation and the differentiation block of these malignant cells. In addition, BRD4 has been identified as a critical sensitivity determinant in a genetically defined AML mouse model (Nature 2011 478(7370):524-8). Suppression of BRD4 led to robust anti-leukemic effects in vitro and in vivo, accompanied by terminal myeloid differentiation. Interestingly, BRD4 inhibition triggered MYC down-regulation in a broad array of mouse and human leukemia cell lines examined, indicating that small molecule BRD4 inhibitors may provide a means to suppress the MYC pathway in a range of AML subtypes. Finally, the other family members of the BET family have also been reported to have some function in controlling or executing aspects of the cell cycle, and have been shown to remain in complex with chromosomes during cell division-suggesting a role in the maintenance of epigenetic memory (Mol. Cell. 2008 30 (1):51-60).
Recent studies indicated that BRD4 expression was upregulated in clinical urothelial carcinoma of the bladder (UCB) tissues, and high expression of BRD4 was associated closely with a more malignant clinical feature and poor prognosis of UCB patients. These results suggested that BRD4 over-expression might be useful as a prognostic factor for UCB patients and that BRD4 inhibition could be a new approach for effective therapy of human UCB (Int J Clin Exp Pathol. 2014; 7(7): 4231-4238).
In the activated B-cell-like (ABC) subtype of diffuse large B-cell lymphoma (DLBCL), NF-κB activity is essential for viability of the malignant cells and is sustained by constitutive activity of IκB kinase (IKK) in the cytoplasm. An unexpected role for the bromodomain and extraterminal domain (BET) proteins BRD2 and BRD4 in maintaining oncogenic IKK activity in ABC DLBCL has been reported (Proc Natl Acad Sci USA. 2014; 111(31):11365-70). IKK activity was reduced by small molecules targeting BET proteins as well as by genetic knockdown of BRD2 and BRD4 expression, thereby inhibiting downstream NF-κB-driven transcriptional programs and killing ABC DLBCL cells. Using a high-throughput platform to screen for drug-drug synergy, it was observed that the BET inhibitor JQ1 combined favorably with multiple drugs targeting B-cell receptor signaling, one pathway that activates IKK in ABC DLBCL.
The BTK kinase inhibitor ibrutinib, which is in clinical development for the treatment of ABC DLBCL, synergized strongly with BET inhibitors in killing ABC DLBCL cells in vitro and in a xenograft mouse model. These findings provide a mechanistic basis for the clinical development of BET protein inhibitors in ABC DLBCL, particularly in combination with other modulators of oncogenic IKK signaling.
Histone acetylation levels have been associated with an open chromatin architecture and transcriptional activation, but specific marks have been linked to chromatin condensation, regulation of metabolism and DNA repair. Therefore, inappropriate acetylation levels have been associated with an aberrant transcription of disease-promoting genes, including cancer-related genes (Oncotarget. 2015; 6(8): 5501-5506).
BET family proteins have been also reported to be involved in a variety of malignant tumors, between which NUT-NMC have been reported to closely related to BRD proteins, in which the BRD-NUT blocks cellular differentiation, and depletion of this oncogene in squamous differentiation and cell cycle arrest. NUT midline carcinoma (NMC), an aggressive squamous cell carcinoma, is accordance with acquired chromosomal rearrangements involving NUT, creating chimeric genes that encode fusion proteins. Usually BRD4-NUT fusion genes are been detected, and less commonly NUT-variant fusion genes involving BRD3 also exists, leading to the expression of BRD-NUT fusion proteins (Expert Rev Mol Med. 2011; 13: e29).
In addition, BET family proteins have reported to able to promote aberrant gene expression in leukemia. MYC family transcription factors are key regulators of cell growth and survival, whose gene amplification is a common copy-number alteration in cancer, while overexpression or translocation of the MYC locus contributes to Myc activity deregulation. In hematologic cancer models, such as MLL-fusion leukemia [46], acute myeloid leukemia (AML) (Nature 2011; 478: 524-528), Burkitt's lymphoma (Proc. Natl. Acad. Sci. USA 2011; 108: 16669-16674), multiple myeloma (Cell. 2011; 146: 904-917), and B-cell acute lymphoblastic (BLL) leukemia (Blood. 2012; 120:2843-2852), amplification of onco-protein Myc drivesm distinct transcription programs, and leads to a consequence of cell proliferation. BET family directly regulate the expression of MYC genes, and directly silencing MYC gene expression via disruption of BET protein binding at the MYC locus may largely reduce cell proliferation (Nature 2010; 463: 899-905).
Androgen receptor (AR) signalling is typically reactivated in patients with castration-resistant prostate cancer (CRPC). However, acquired resistance to therapies that target AR signalling often occurs, so there is a need to identify additional therapeutic targets within this signalling network. Studies have found that the bromodomain and extraterminal (BET) family of chromatin readers might be one such target. Treatment of a panel of prostate cancer cell lines with the small molecule BET inhibitor JQ1 revealed that those with activated AR signaling were sensitive to JQ1-induced apoptosis and cell cycle arrest, and this was phenocopied by knockdown of bromodomain-containing protein 2 (BRD2), BRD3 or BRD4, which are all BET family proteins. JQ1 also globally reduced the levels of transcription of AR target genes in AR-positive cells, which suggests that BET family proteins are involved in AR-mediated transcriptional programmes.
Interestingly, in prostate cell lines, which have both AR amplification and the transmembrane protease, serine 2 (TMPRSS2)-ETS-related gene (ERG) fusion gene, the expression of ERG and the ERG transcriptional programme were blocked by bromodomain inhibition through the reduction of AR and BRD4 binding to the TMPRSS2 promoter/enhancer.
Evidence suggests that JQ1 functions downstream of AR, therefore it may be effective in the context of acquired resistance mediated by AR activation (for example, by AR amplification or mutation), which is common in patients with CRPC (Nature 2014; 510(7504):278-82).
In another study, BRD2 and BRD4 RNA were found to be significantly overexpressed in Glioblastoma Multiforme (GBM), suggesting that BET protein inhibition may be an effective means of reducing GBM cell proliferation. Disruption of BRD4 expression in glioblastoma cells reduced cell cycle progression. Similarly, treatment with the BET protein inhibitor I-BET151 reduced GBM cell proliferation in vitro and in vivo. BET inhibition treatment enriched cells at the G1/S cell cycle transition. Importantly, BET inhibitors were as potent at inhibiting GBM cell proliferation as temozolomide (TMZ), the current chemotherapy treatment administered to GBM patients. Since BET inhibitors inhibit GBM cell proliferation by arresting cell cycle progression, suggests that BET protein inhibition as a viable therapeutic option for GBM patients suffering from TMZ resistant tumors (Epigenetics. 2014; 9(4):611-20).
Recent studies have unraveled a possible mechanism about how BRD4 proteins are involved in the transcription of active genes in cancers, especially associated with a subset of these genes. As previous mentioned, BRD4 and Mediator form a complex in transcription process, this complex may related to super-enhancers, which are span large genomic regions and contain exceptional amounts of Mediator and BRD4 (Oncotarget. 2015; 6(8): 5501-5506).
In addition, important tumor genes are also associated with super-enhancers, so far has been identified in myeloma, small-cell lung cancer and glioblastoma. Therefore, BRD4 may regulate ongenetic drivers, such as MYC, through occupying super-enhancers, while inhibition of BRD4 also leads to preferential disruption of super-enhancers and selective loss of oncogene expression (Cell. 2013; 153(2): 320-334).
Furthermore, Cancers of neural origin may be related to the distinct expression of BET proteins including glioblastoma, medulloblastoma, and neuroblastoma. For instance, neuroblastoma is a pediatric solid tumor associated with a high frequency of MYCN amplifications, and inhibition of BET proteins in neuroblastoma leads to cell arrest (Cancer Discov. 2013; 3:308-323). BET proteins are also required for glioblastoma cell proliferation, mRNA of BRD2 and BRD4 are significantly overexpressed in glioblastoma, while disruption of BRD4 expression reduced glioblastoma cell cycle progression (Epigenetics. 2014; 9: 611-620).
Also, in melanoma, tumor progression may contribute to epigenetic changes, thus epigenetic and/or transcriptional regulation of certain target genes may support melanoma tumor-genesis. NF-κB regulates cytokine and chemokine production in melanoma, and is believed to contribute to progression of the disease by up-regulation of cell cycle and anti-apoptotic genes. BRD2 and BRD4 are overexpressed in human primary and metastatic melanomas, whose inhibition resulted in down-regulating production of cytokines such as IL-6 and IL-8 (Ann Rheum Dis. 2014; pii: annrheumdis-2014-205809).
Furthermore, the largely hydrophobic nature of the central acetylated lysine binding pocket and special extra-terminal domain of BET are necessary to accommodate the charge neutralized acetylated lysine and recruits related proteins, making these modules particularly attractive for the development of inhibitors. Many proteins that use BRDs for their recruitment to specific regulatory complexes have been implicated in the development of cancer (Nature Reviews Drug Discovery, 2014, 13(5): 337-356).
Studies on the transcriptional effects of BET inhibitors demonstrated that the inhibition of BET bromodomains selectively interfered with gene expression programmes that mediated cellular growth and evasion of apoptosis in cancers. Given the general role of BET proteins in transcriptional elongation, the discovery that inhibition of these proteins only affects the transcription of a small subset of genes was unexpected and suggested that inhibitors of bromodomains may specifically modulate the expression of some disease-promoting genes. Examples of bromo domain inhibitors include benzodiazepine derivatives, disclosed in WO2011/054553, and imidazo quinolone derivatives, disclosed in WO20111054846. Thus, there is the need to provide BRD inhibitors and especially BRD4 inhibitors for use in the prevention and/or treatment of diseases characterized by excessive or abnormal cell proliferation, such as cancer.
Although much attention has been focused on the oncologic applications of the bromodomain inhibitors, bromodomain inhibitors have shown inhibition against T-cell mediated inflammation in a rat model at a level comparable to that of the positive control compound, rapamycin.
In addition some viruses make use of BET proteins to tether their genomes to the host cell chromatin, as part of the process of viral replication (Cell 2004 117(3):349-60). Therefore, BET inhibition may have a broad range of therapeutic applications beyond oncology, such as inflammation, heart failure, and male contraception [Cell 2013, 154 (3), 569-582; Cell 2012, 150 (4), 673-684].
Fluorine has found interest in bioorganic and structural chemistry over the past decade and has become a useful feature in small molecule drug design. The small and highly electronegative fluorine atom can play a useful role in medicinal chemistry. Selective installation of fluorine into a therapeutic or diagnostic small molecule candidate can provide a number of favorable pharmacokinetic and/or physicochemical properties including improved metabolic stability and enhanced membrane permeation. Increased binding affinity of fluorinated drug candidates to a target protein has also been documented in some cases. A further emerging application of the fluorine atom is the use of 18F isotope as a radiolabel tracer atom in the sensitive technique of Positron Emission Tomography (PET) imaging.
Fluorine substitution has been investigated in drug research as a means of enhancing biological activity and/or increasing chemical and/or metabolic stability. Factors to be considered when synthesising fluorine-containing compounds include (a) the relatively small size of the fluorine atom (van der Waals radius of 1.47 Å), comparable to hydrogen (van der Waals radius of 1.20 Å), (b) the highly electron-withdrawing nature of fluorine, (c) the greater stability of the C—F bond compared to the C—H bond, and (d) the greater lipophilicity of fluorine compared to hydrogen.
Despite the fact that fluorine is slightly larger than hydrogen, several studies have shown that the fluorine atom is a reasonable hydrogen mimic with minimal steric perturbations with respect to the compound's mode of binding to a receptor or enzyme (Annu. Rev. Pharmacol. Toxicol. 2001, 41, 443-470). However, the introduction of a fluorine atom can significantly alter the physicochemical properties of a compound due to its high electronegativity. Therefore, this type of modification can potentially induce altered biological responses of the molecule.